Acute, infectious diarrhea is a leading cause of disease and death in many areas of the world. In developing countries, the impact of diarrheal disease is staggering. For Asia, Africa and Latin America, it has been estimated that there are between 3-5 billion cases of diarrhea each year and of those cases, about 5-10 million result in death. Walsh, J. A. et al.: N. Engl. J. Med., 301:967-974 (1979).
Rotaviruses have been recognized as one of the most important causes of severe diarrhea in infants and young children since their discovery in 973. It is estimated that rotavirus disease is responsible for over one million deaths annually. Rotavirus-induced illness most commonly affects children between 6 and 24 months of age, and the peak prevalence of the disease generally occurs during the cooler months in temperate climates, and year-round in tropical areas. Rotaviruses are typically transmitted from person to person by the fecal-oral route with an incubation period of from about 1 to about 3 days. Unlike infection in the 6-month to 24-month age group, neonates are generally asymptomatic or have only mild disease. In contrast to the severe disease normally encountered in young children, most adult rotavirus infections are mild or asymptomatic because such episodes represent reinfection generally as a result of contact with children known to be excreting rotavirus. Offit, P. A. et al.: Comp. Ther., 8(8):21-26 (1982).
Rotaviruses are generally spherical, and their name is derived from their distinctive outer and inner or double-shelled capsid structure. The outer capsid has a diameter of about 70 nm, whereas the inner capsid has a diameter of about 55 nm. Flewett, T. H. et al.: J. Clin. Path., 27:603-614 (1974). Typically, the double-shelled capsid structure of a rotavirus surrounds an inner protein shell or core which contains the genome. The genome of a rotavirus is composed of 11 segments of double-stranded RNA which encode at least 11 distinct viral proteins. Two of these viral proteins designated as VP4 and VP7 are believed to be arranged on the exterior of the double-shelled capsid structure. The inner core of the rotavirus presents one protein, i.e., rotavirus protein designated as VP6. The relative importance of these three particular rotaviral proteins in eliciting the immune response that follows rotavirus infection is not yet clear. Nevertheless, VP6 protein constitutes the group and subgroup antigen, and VP4 and VP7 proteins are the determinates of serotype specificity.
VP7 protein is believed to be a 38,000 MW glycoprotein which is the translational product of genomic segment 7, 8 or 9. This protein is believed to stimulate formation of the major neutralizing antibody following rotavirus infection. VP4 protein is believed to be a non-glycosylated protein of approximately 88,000 MW which is the translational product of genomic segment 4. This protein is also believed to stimulate neutralizing antibody following rotavirus infection.
Since VP4 and VP7 proteins are the viral proteins against which neutralizing antibodies are directed, they are believed to be prime candidates for development of rotavirus vaccines affording protection against rotavirus illness. However, cells infected with rotavirus do not secrete VP4 and VP7 proteins and, consequently, the immune system is likely to see these proteins only when the cells lyse and the rotaviruses are released.
Human rotaviruses may be divided into six serotypes, i.e., serotypes 1-4 and 8-9, based upon differences in the VP4 and VP7 proteins. Human rotaviruses may also be divided into two subgroups, i.e., subgroups I and II, based on antigenic differences in the inner capsid VP6 protein. Serotype 2 and 8 strains of human rotavirus usually belong to subgroup I, and serotypes 1, 3, 4 and 9 of human rotavirus strains usually belong to subgroup II. Most diseases are presently believed to be caused by rotaviruses belonging to serotypes 1-4 strains and serotype 1 strains have been found to predominate in many geographic regions.
In addition to subgrouping and serotyping, human rotaviruses have been broadly classified into two additional groups, i.e., those with either "short" or "long" RNA patterns based on mobility of their RNA genome segments during gel electrophoresis. Subgroup I human rotavirus strains typically have characteristic "short" electrophoretic patterns whereas subgroup II human rotavirus strains typically have characteristic "long" electrophoretic patterns. The "short" patterns of rotavirus RNA have been associated with an inverse in migration order of gene segments 10 and 11. The majority of rotavirus strains which have been isolated up to now, however, are those with the characteristic "long" patterns.
The human rotaviruses that cause severe diarrhea were originally divided into two "genogroups" based on a genetic linkage between all 11 genomic segments. In 1982, however, a first natural human rotavirus strain in which this linkage appeared to have been broken was isolated. This human rotavirus strain was designated as AU-1. Since that time, a number of human rotaviruses with properties similar to those of the AU-1 human rotavirus strain have been reported in Aboudy, Y. I. et al.: J. Med. Virol., 25:351-359 (1988); Beards, G. M. et al.: J. Clin. Microbiol., 27:2827-2833 (1989); Brown, D. W. G. et al: J. Clin. Microbiol., 26:2410-2414 (1988); Gosh, S. K. et al.: Arch. Virol., 105:119-127 (1989); Sethi, S. K. et al.: J. Med. Virol., 26:249-259 (1988); and Steele, A. D. et al.: J. Med. Virol., 24:321-327 (1988). These human rotaviruses belong to subgroup I, but are identified as serotype 3 and have " long" electrophoretic patterns, properties common to many animal rotavirus strains. Thus, three human rotavirus genogroups have been defined heretofore based on genetic homology of the prototype strains designated as Wa (subgroup II, "long" electropherotype), DS-1 (subgroup I, "short" electropherotype) and AU-1 (subgroup I, "long" electropherotype). See U.S. Pat. Nos. 4,853,333 and 4,341,870 which are believed to be relative to the Wa human rotavirus serotype 1 subgroup II strain.
Because human rotaviruses are formed with segmented genomes, co-infection of cells with two distinct strains of rotavirus can result in the formation of rotavirus progeny having gene segments inherited from both parental strains. A presumed natural reassortant between subgroup II strains was reported some years ago, but natural reassortants between subgroups I and II human rotaviruses have not as of yet been isolated or identified in Hoshino, Y. et al.: Proc. Natl. Acad. Sci. USA, 82:8701-8704 (1985). Recently, however, human rotaviruses having atypical associations between subgroup, serotype and electropherotypes have been reported in Ahmed, M. U. et al.: J. Clin. Microbiol., 27:1678-1681 (1987); Beards, G. M. et al.: J. Clin. Microbiol., 27:2827-2833 (1989); Mascarenhas, J. D. P. et al.: Virus Res., 14:235-240 (1989); Matsuno, S. et al.: Virus Res., 10:167-175 (1988); Steele, A. D. et al.: J. Med. Virol., 24:321-327 (1988). See also U.S. Pat. No. 4,571,385 which apparently discloses a reassortant rotavirus derived from the human rotavirus strain D, serotype 1 and the bovine rotavirus UK strain.
In view of the seriousness of rotavirus illness and that present therapy is limited to non-specific supportive measures, such as replacement of fluids and electrolytes, the development of an effective rotavirus vaccine that offers complete protection against all serotypes of human rotavirus is an important priority for health care in the United States and in developing countries. Several approaches to the development of a rotavirus vaccine have been pursued. Two approaches that have been evaluated in humans include the use of a live, attenuated human rotavirus strain, and the use of a rotavirus strain of animal origin.
Initial rotavirus vaccine studies suggested that bovine rotavirus strains offered partial protection to infants against heterotypic human rotaviruses even in the absence of detectable neutralizing antibody to circulating human strains. See Vesikari, T. et al.: Lancet, 2:807-311 (1983); Vesikari, T. et al.: Lancet, 1:977-981 (1984); Vesikari, T. et al.: J. Pediatr., 107:189-194 (1985); and Clark, H. F. et al.: J. Infect. Dis., 158:570-587 (1988). Subsequent efficacy trials, however, indicated that these bovine rotavirus vaccines are, at best, only marginally protective. See Hanlon, P. et al.: Lancet, 1:1342-1345 (1987); DeMol, P. et al.: Lancet, 2:108 (1986); and Lanata, C.G. et al.: J. Infect. Dis., 159:452-459 (1989). For example, the RIT 4237 bovine strain rotavirus (serotype 6) appeared to be successful in preventing clinically significant diarrhea due to rotavirus infection in Finish infants. See Vesikari, T. et al.: Lancet, 2:807-811 (1983) and Vesikari, T. et al.: Lancet, 1:977-981 (1984). When later evaluated in developing countries, however, it was not as effective. See Hanlon, P. et al.: Lancet, 1:1342-1345 (1987); and DeMol, P. et al.: Lancet, 108 (1986). See also, U.S. Pat. Nos. 4,636,385; 4,341,763; and 4,190,645.
Rhesus rotavirus (RRV) vaccine strain MMU18006 (serotype 3) has been shown to be immunogenic in several studies, Losonky, G. A. et al.: Ped. Infect. Dis., 5:25-29 (1986); Vesikari, T. et al.: J. Infect. Dis., 153:832-839 (1986); Anderson, E. L. et al.: J. Infect. Dis., 153:823-831 (1986); Gothefors, L. et al.: J. Infect. Dis., 159:753-757 (1989); and Wright, P. F. et al.: Pediatrics, 80:473-480 (1987), but has been associated with mild side effects including low grade fever and watery stools. Losonky, G. A. et al.: Ped. Infect. Dis., 5:25-29 (1986); Vesikari, T. et al.: J. Infect. Dis., 153:832-839 (1986); Anderson, E. L. et al.: J. Infect. Dis., 153:823-831 (1986); Gothefors, L. et al.: J. Infect. Dis., 159:753-757 (1989). Clinical evaluations involving RRV vaccine have revealed protection against severe diarrhea in studies conducted in Venezuela, Flores, J. et al.: Lancet, 1:882-884 (1987), and Sweden, Gothefors, L. et al.: J. Infect. Dis., 159:753-757 (1989), but no protection in a study of Navajo Indian children. See Santosham, M. et al.: A Field of Study of the Safety and Efficacy of Two Candidate Rotavirus Vaccines Program and Abstracts: 27th Conference on Antimicrobial Agents and Chemotherapy (New York), 99 (1987). Thus, efficacy trials with RRV indicated that this vaccine strain protected against homotypic serotype 3 human rotaviruses, but not heterotypic human strains. See Flores, J. et al: J. Clin. Microbiol., 27:512-518 (1989). Moreover, results from other studies conducted involving this rhesus rotavirus vaccine have varied. See Christy, C. et al.: Pediatr. Res., 25:157A (1989). See also U.S. Pat. Nos. 4,751,080 and 4,704,275.
Serotype-specific protection has also been reported after natural rotavirus infection and is correlated with the titer of neutralizing antibody present in the previously infected humans. See Chiba, S. et al: Lancet, 2:417-421 (1986). Thus, serotypespecific neutralizing antibody may be the primary determinate of protection against rotavirus disease. Consequently, because there are at least six serotypes of human rotaviruses, a mono-serotype or heterotypic (animal) vaccine may be insufficient to provide complete protection against all serotypes associated with human rotavirus disease.
It has also been reported that primary rotavirus infection in both humans and animals usually result in production of neutralizing antibody to one predominant rotavirus serotype, although some neutralizing antibody to rotaviruses belonging to other serotypes is often detected. See Gerna, G. et al.: Infect. Immun., 43:722-729 (1984); Snodgrass, D. R. et al.: J. Clin. Microbiol., 20:342-346 (1984); Clark, H. F. et al.: Ped. Infect. Dis., 4:626-631 (1985); Puerto, F. I. et al.: J. Clin. Microbiol., 25:960-963 (1987); Zheng, B. J. et al.: J. Clin. Microbiol., 26:1506-1512 (1988); and Brussow, H. et al.: J. Infect. Dis., 158:588-595 (1988). Subsequent rotavirus infection or inoculation with different serotypes has resulted in neutralizing antibody production to the new virus strain and an increase in pre-existing antibody titers to the other rotavirus serotypes involved in previous infection, presumably due to an amnestic response. See Clark, H. F. et al.: Ped. Infect. Dis., 4:626-631 (1985); Kapikan, A. Z. et al.: J. Infect. Dis., 147:95-106 (1983); Urasawa, S. et al.: Arch. Virol., 81:1-12 (1984); Ward, R. L. et al.: J. Infect. Dis., 154:871-880 (1986); Woode, G. N. et al.: J. Clin. Microbiol., 25:1052-1058 (1987); Brussow, H. et al.: J. Gen. Virol., 69:1647-1658 (1988); and Bernstein, D.I. et al.: Antiviral Res., 12:293-300 (1989). These studies have not, however, evaluated the ability of vaccine strains of rotavirus to boost neutralizing antibody titers to heterotypic strains of human rotavirus in infants with pre-existing antibody due to a previous infection. They also have not determined whether previous vaccination with a heterotypic strain can broaden or increase neutralizing antibody responses to human rotaviruses during subsequent natural infection. Notwithstanding, it has been shown that when children are vaccinated with the same rotavirus serotype as the strains causing natural infections, the neutralizing antibody produced after infection prevents "take" of the vaccine at the time of revaccination. See Tajma, T. et al.: Vaccine, 8:70-74 (1990).
Consequently, there is a serious need for a rotavirus vaccine which will provide effective immunity against human rotaviruses belonging to at least serotypes 1-4, and in particular serotype 1 which is believed to be the most predominant serotype of human rotavirus.